STRUCTURAL STUDIES OF NUCLEOTIDE DEPENDENT ENZYMES

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DESCRIPTION: Nucleotides serve important roles in virtually all biochemical processes where they provide building blocks for DNA and RNA, act as currencies of energy in various metabolic pathways, serve as modulators of enzymatic activity, and function as transient electron acceptors and donors. The objective of this research program is to understand, on a molecular level, the role of nucleotides in the overall catalytic mechanisms of four protein systems: acetyl-CoA carboxylase which is Mg(II)-ATP dependent, kanamycin nucleotidyltransferase which can employ ATP, GTP, or UTP as a substrate, UDP-galactose 4-epimerase which requires tightly bound NAD for activity, and L-phenylalanine dehydrogenase which binds NAD or NADH reversibly. Kanamycin nucleotidyltransferase catalyzes the inactivation of various aminoglycoside antibiotics often employed in the treatment of serious infections due to aerobic Gram-negative bacteria. UDP-galactose 4- epimerase plays a key role in proper galactose metabolism, and indeed, one form of galactosemia arises from a deficiency in this enzyme. Acetyl-CoA carboxylase catalyses the first and absolutely critical step in the biosynthesis of long chain fatty acids. Finally, L-phenylalanine dehydrogenase catalyzes the oxidative deamination of L-phenylalanine. For the proposed studies, a combination of site-directed mutagenesis, x- ray crystallography, and kinetic measurements will be employed. Taken together, these studies will provide a detailed description of the role of nucleotides in the enzymatic functions of these proteins.